Pricing Comment

We are happy to accommodate requests for single genes or a subset of these genes. The price will remain the list price. If desired, free reflex testing to remaining genes on panel is available. Alternatively, a single gene or subset of genes can also be ordered on our PGxome Custom Panel.

Pricing Comment

Turnaround Time

Clinical Sensitivity

Thus far, no gross deletions or duplications have been reported in SCN1B (Human Gene Mutation Database).

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Clinical Features

Generalized (or genetic) epilepsy with febrile seizures plus (GEFS+) is a familial epilepsy syndrome characterized by frequent episodes of febrile seizures beyond the age of 5 years. Afebrile seizures also occur. Patients with GEFS+ usually have at least two family members with GEFS+ spectrum phenotypes, and seizure-type heterogeneity is typical among family members (Scheffer et al. 2009). Although generalized epilepsies predominate in GEFS+ families, focal or partial epilepsies are also recognized with and without preceding febrile seizures. This disorder usually has a good prognosis (Wilmshurst et al. 2015).

Genetics

This panel for GEFS+ contains 4 voltage-gated sodium channel genes (SCN1A, SCN1B, SCN2A, SCN9A) and 1 ligand-gated neurotransmitter receptor subunit gene (GABRG2). Generalized epilepsy with febrile seizures plus is inherited in an autosomal dominant manner with incomplete penetrance. GEFS+ cases can also be sporadic with no family history. Pathogenic variants in SCN1A, SCN1B, SCN2A, GABRG2 have been well documented to cause GEFS+ (Ottman et al. 2010; Wilmshurst et al. 2015). In addition, SCN9A was also reported to be associated with cases of GEFS+ (Mulley et al. 2013; Singh et al. 2009). Each gene in this panel is discussed briefly below: SCN1A: encodes an alpha subunit of the voltage-gated sodium channel. It is proposed that pathogenic variants in SCN1A relieve GABAergic inhibition in the neocortex and hypothalamus, resulting in neuronal hyperactivity and epilepsy (Yu et al. 2006; Catterall et al. 2010). Missense variants in SCN1A can cause GEFS+ (Guerrini et al. 2010). SCN1B: encodes a beta subunit of the voltage-gated sodium channel. SCN1B has been shown to modulate the excitability of sodium channels, mediate cell-cell adhesions and direct neurite outgrowth- all of which may contribute to the epilepsy phenotypes associated with SCN1B pathogenic variants (Patino et al. 2009). Pathogenic variants in SCN1B have been identified in a small percent of GEFS+ families and show ~63% penetrance (Scheffer et al. 2007; Audenaert et al. 2003). SCN2A: encodes an alpha subunit of the voltage-gated sodium channel. Sodium channels are excitatory and propagate action potentials in neurons. The SCN2A channel is sensitive to temperature changes, which likely contributes to the genesis of febrile seizures. SCN2A gain-of function pathogenic variants cause GEFS+ (Sugawara et al. 2001; Liao et al. 2010). GABRG2: encodes a gamma subunit of the GABA-A receptor. GABA is an inhibitory neurotransmitter responsible for modulating signaling in the brain. It has been proposed that the loss of inhibition in neurons results in increased synaptic excitation and epilepsy (Lachance-Touchette et al. 2011). Certain pathogenic variants in GABRG2 are especially sensitive to elevated temperature, hence their association with febrile seizures (Kang et al. 2006). Both missense and nonsense variants in GABRG2 have been reported in cases of GEFS+. The penetrance of GABRG2 pathogenic variants is incomplete (Lachance-Touchette et al. 2011). SCN9A: encodes the alpha subunit of the voltage-gated sodium channel. Heterozygous gain-of-function missense variants in SCN9A cause GEFS+, type 7. The phenotype progresses to febrile and afebrile seizures and, in some patients, epilepsy (Singh et al. 2009; Singh et al. 1999).

Testing Strategy

For this Next Generation (NextGen) panel, the full coding regions plus ~10 bp of non-coding DNA flanking each exon are sequenced for each of the genes listed below. Sequencing is accomplished by capturing specific regions with an optimized solution-based hybridization kit, followed by massively parallel sequencing of the captured DNA fragments. Additional Sanger sequencing is performed for any regions not captured or with insufficient number of sequence reads. All pathogenic and undocumented variants are confirmed by Sanger sequencing.

Indications for Test

Candidates for this panel include patients with symptoms suspected for generalized epilepsy with febrile seizures plus. This panel is made based on the recent ILEA recommendation for the management of infantile seizures and especially aids in a differential diagnosis of similar phenotypes by analyzing multiple genes simultaneously (Wilmshurst et al. 2015).

TEST METHODS

NextGen Sequencing using PG-Select Capture Probes

Test Procedure

We use a combination of Next Generation Sequencing (NGS) and Sanger sequencing technologies to cover the full coding regions of the listed genes plus ~20 bases of non-coding DNA flanking each exon. As required, genomic DNA is extracted from the patient specimen. For NGS, patient DNA corresponding to these regions is captured using an optimized set of DNA hybridization probes. Captured DNA is sequenced using Illumina’s Reversible Dye Terminator (RDT) platform (Illumina, San Diego, CA, USA). Regions with insufficient coverage by NGS are covered by Sanger sequencing. All pathogenic, likely pathogenic, or variants of uncertain significance are confirmed by Sanger sequencing.

For Sanger sequencing, Polymerase Chain Reaction (PCR) is used to amplify targeted regions. After purification of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit. PCR products are resolved by electrophoresis on an ABI 3730xl capillary sequencer. In nearly all cases, cycle sequencing is performed separately in both the forward and reverse directions.

Patient DNA sequence is aligned to the genomic reference sequence for the indicated gene region(s). All differences from the reference sequences (sequence variants) are assigned to one of five interpretation categories, listed below, per ACMG Guidelines (Richards et al. 2015).

Human Genome Variation Society (HGVS) recommendations are used to describe sequence variants (http://www.hgvs.org). Rare variants and undocumented variants are nearly always classified as likely benign if there is no indication that they alter protein sequence or disrupt splicing.

Analytical Validity

As of March 2016, 6.36 Mb of sequence (83 genes, 1557 exons) generated in our lab was compared between Sanger and NextGen methodologies. We detected no differences between the two methods. The comparison involved 6400 total sequence variants (differences from the reference sequences). Of these, 6144 were nucleotide substitutions and 256 were insertions or deletions. About 65% of the variants were heterozygous and 35% homozygous. The insertions and deletions ranged in length from 1 to over 100 nucleotides.

In silico validation of insertions and deletions in 20 replicates of 5 genes was also performed. The validation included insertions and deletions of lengths between 1 and 100 nucleotides. Insertions tested in silico: 2200 between 1 and 5 nucleotides, 625 between 6 and 10 nucleotides, 29 between 11 and 20 nucleotides, 25 between 21 and 49 nucleotides, and 23 at or greater than 50 nucleotides, with the largest at 98 nucleotides. All insertions were detected. Deletions tested in silico: 1813 between 1 and 5 nucleotides, 97 between 6 and 10 nucleotides, 32 between 11 and 20 nucleotides, 20 between 21 and 49 nucleotides, and 39 at or greater than 50 nucleotides, with the largest at 96 nucleotides. All deletions less than 50 nucleotides in length were detected, 13 greater than 50 nucleotides in length were missed. Our standard NextGen sequence variant calling algorithms are generally not capable of detecting insertions (duplications) or heterozygous deletions greater than 100 nucleotides. Large homozygous deletions appear to be detectable.

Analytical Limitations

Interpretation of the test results is limited by the information that is currently available. Better interpretation should be possible in the future as more data and knowledge about human genetics and this specific disorder are accumulated.

When Sanger sequencing does not reveal any difference from the reference sequence, or when a sequence variant is homozygous, we cannot be certain that we were able to detect both patient alleles. Occasionally, a patient may carry an allele which does not amplify, due to a large deletion or insertion. In these cases, the report will contain no information about the second allele. Our Sanger and NGS Sequencing tests are generally not capable of detecting Copy Number Variants (CNVs).

We sequence all coding exons for each given transcript, plus ~20 bp of flanking non-coding DNA for each exon. Test reports contain no information about other portions of the gene, such as regulatory domains, deep intronic regions or any currently uncharacterized alternative exons.

In most cases, we are unable to determine the phase of sequence variants. In particular, when we find two likely causative mutations for recessive disorders, we cannot be certain that the mutations are on different alleles.

Our ability to detect minor sequence variants due to somatic mosaicism is limited. Sequence variants that are present in less than 50% of the patient’s nucleated cells may not be detected.

Runs of mononucleotide repeats (eg (A)n or (T)n) with n >8 in the reference sequence are generally not analyzed because of strand slippage during PCR.

Unless otherwise indicated, DNA sequence data is obtained from a specific cell-type (usually leukocytes from whole blood). Test reports contain no information about the DNA sequence in other cell-types.

We cannot be certain that the reference sequences are correct.

Rare, low probability interpretations of sequencing results, such as for example the occurrence of de novo mutations in recessive disorders, are generally not included in the reports.

We have confidence in our ability to track a specimen once it has been received by PreventionGenetics. However, we take no responsibility for any specimen labeling errors that occur before the sample arrives at PreventionGenetics.

Test Procedure

Equal amounts of genomic DNA from the patient and a gender matched reference sample are amplified and labeled with Cy3 and Cy5 dyes, respectively. To prevent any sample cross contamination, a unique sample tracking control is added into each patient sample. Each labeled patient product is then purified, quantified, and combined with the same amount of reference product. The combined sample is loaded onto the designed array and hybridized for at least 22-42 hours at 65°C. Arrays are then washed and scanned immediately with 2.5 µM resolution. Only data for the gene(s) of interest for each patient are extracted and analyzed.

Analytical Validity

PreventionGenetics' high density gene-centric custom designed aCGH enables the detection of relatively small deletions and duplications within a single exon of a given gene or deletions and duplications encompassing the entire gene. PreventionGenetics has established and verified this test's accuracy and precision.

Analytical Limitations

Our dense probe coverage may allow detection of deletions/duplications down to 100 bp; however due to limitations and probe spacing this cannot be guaranteed across all exons of all genes. Therefore, some copy number changes smaller than 100-300 bp within a targeted large exon may not be detected by our array.

This array may not detect deletions and duplications present at low levels of mosaicism or those present in genes that have pseudogene copies or repeats elsewhere in the genome.

aCGH will not detect balanced translocations, inversions, or point mutations that may be responsible for the clinical phenotype.

Breakpoints, if occurring outside the targeted gene, may be hard to define.

The sensitivity of this assay may be reduced when DNA is extracted by an outside laboratory.

Ship blood tubes at room temperature in an insulated container. Do not freeze blood.

During hot weather, include a frozen ice pack in the shipping container.
Place a paper towel or other thin material between the ice pack and the blood tube.

In cold weather, include an unfrozen ice pack in the shipping container as insulation.

At room temperature, blood specimen is stable for up to 48 hours.

If refrigerated, blood specimen is stable for up to one week.

Label the tube with the patient name, date of birth and/or ID number.

DNA

(Delivery accepted Monday - Saturday)

Send in screw cap tube at least 5 µg -10 µg of purified DNA at a concentration of at least 20 µg/ml for NGS and Sanger tests and at least 5 µg of purified DNA at a concentration of at least 100 µg/ml for gene-centric aCGH, MLPA, and CMA tests, minimum 2 µg for limited specimens.

For requests requiring more than one test, send an additional 5 µg DNA per test ordered when possible.

DNA may be shipped at room temperature.

Label the tube with the composition of the solute, DNA concentration as well as the patient’s name, date of birth, and/or ID number.

We only accept genomic DNA for testing. We do NOT accept products of whole genome amplification reactions or other amplification reactions.

CELL CULTURE

(Delivery preferred Monday - Thursday)

PreventionGenetics should be notified in advance of arrival of a cell culture.

Culture and send at least two T25 flasks of confluent cells.

Some panels may require additional flasks (dependent on size of genes, amount of Sanger sequencing required, etc.). Multiple test requests may also require additional flasks. Please contact us for details.

Send specimens in insulated, shatterproof container overnight.

Cell cultures may be shipped at room temperature or refrigerated.

Label the flasks with the patient name, date of birth, and/or ID number.

We strongly recommend maintaining a local back-up culture. We do not culture cells.